Combination therapy to enhance NK cell mediated cytotoxicity

ABSTRACT

The function of natural killer (NK) cells is regulated by inhibitory and activating signals delivered by cell surface receptors, 1-7F9 is a fully human monoclonal antibody (mAb) directed against KIR2DL1 and KIR2DL2/3 receptors that block its interaction with its HLA-C ligands breaking NK cell tolerance to autologous tumor cells. Lenalidomide has been shown to increase NK cell cytotoxicity in vitro. The combination of lenalidomide and 1-7F9 enhanced NK cell mediated cytotoxicity against U266 cells beyond that observed with each agent alone. Lenalidomide also increased the expression of NKG2D, DNAM-I and TRAIL ligands including: MICA, ULB P2, CD1 12 and DR 4 on U266 cells. In in vitro cytotoxicity assays, lenalidomide enhanced the susceptibility of myeloma cell lines to NK cell. The NK cell signaling pathways was also explored after lenalidomide treatment and the results show that lenalidomide may upregulate the phospho-SHIP1 (Tyr1020) and has no effect on phosphop44/42 (ERK 1/2) (Thr202/Tyr204) in NK cells. These results provide pre-clinical rationale for clinical investigation of 1-7F9 anti-KIR mAb and lenalidomide in MM.

CROSS-REFERENCE TO RELATED APPLICATION

This application is the U.S. national stage application of InternationalPatent Application No. PCT/US2009/066894, filed Dec. 4, 2009, whichclaims the benefit of U.S. Provisional Patent Application No. 61/120,242filed on Dec. 5, 2008, which are herein incorporated by reference intheir entireties.

The sequence listing in the file named “43271o3801.txt” having a size of6,555 bytes that was created Oct. 14, 2013 is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

This invention relates generally to, methods of enhancing natural killercell mediated cytotoxicity of tumor cells by the combination of anti-KIRmAB (1-7F9) monoclonal antibody and immunomodulatory agents, such aslenalidomide (3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)piperidine-2,6-dione).

BACKGROUND

Natural killer cells (NK cells) are a component of the innate immunesystem. These cells are classified as cytotoxic lymphocytes. These cellsare unique and different to other cells of the immune cells in that theydo not require prior exposure to antigen and do not have receptors forrecognition of specific antigen sequences. Rather, NK cells expressactivating and inhibitory receptors that recognize ligands on targetcells, with activation of NK cells dependent on the balance ofactivating and inhibitory signals mediated by these receptors.Importantly, NK cells express killer immunoglobulin-like receptors (KIR)that are inhibited by Class I major histocompatibility (MHC) markers ontarget cells. Transformed or virally infected cells downregulate MHCclass I molecules and are therefore susceptible to killing by NK cells.In an normally functioning immune system NK cells play a primary role inthe destruction of many types of tumor cells and various human cells ofinfected by viruses. NK cells induce apoptosis in target cells byreleasing various factors in to the target cells that activate the cellsown apoptotic pathway.

Given the recalcitrance of many tumor cells to all known therapies totreat cancer and the adverse side effects associated with even many ofthe best therapies to treat cancer there remains a profound need for newcompounds and methods to treat cancerous tumors. Some aspects of theinvention seek to address these needs.

SUMMARY

Some aspects of the invention include methods of killing cellscomprising the steps of: providing a biologically active amount of3-(4-amino-1-oxo-1,3 dihydro-isoindol-2-yl)piperidine-2,6-dione or apharmaceutically acceptable salt thereof; supplying a biologicallyactive amount of an anti-KIR antibody or biologically active fragmentthereof; and contacting said 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)piperidine-2,6-dione and said anti-KIR antibody orbiologically active fragment thereof, with a cell.

In some embodiment the anti-KIR antibody is 1-7F9, or a biologicallyactive fragment thereof. In some embodiments the cells are cancer cellsincluding for example myeloma cells, lymphoma cells, leukemia cells, andU266 cells.

In some embodiments the biologically active amount of3-(4-amino-1-oxo-1,3 dihydro-isoindol-2-yl)piperidine-2,6-dione is lessthan or equal to 10 μmolL⁻¹. In some embodiments the biologically activeamount of 1-7F9 is equal to or less than 30 ug/ml, or an amount known tosaturate KIR receptors on NK cells. Still other aspects of the inventioninclude methods of treating a patient, comprising the steps of:providing a therapeutically effective amount of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)piperidine-2,6-dione or a pharmaceuticallyacceptable salt thereof; supplying a therapeutically effective amount ofan anti-KIR antibody, or a fragment thereof; and administering saidtherapeutically effective amounts of said lenalidomide and anti-KIRantibody to a patient in need thereof. In some embodiments the anti-KIRantibody is the monoclonal antibody 1-7F9. In some embodiments thepatient is either a human being or a non-human animal. In one aspect,the patient to be treated by the above-described method is a patientdiagnosed with cancer, e.g. a myeloma, a leukemia, a lymphoma. In a moreparticular aspect, the patient is a patient diagnosed with multiplemyeloma (MMy). In another particular aspect, the patient is a patientdiagnosed with chronic myeloid leukaemia (CML). In still anotherparticular aspect, the patient is a patient diagnosed with acute myeloidleukaemia (AML). In another particular aspect, the patient is a patientdiagnosed with chronic lymphocytic leukemia (CLL) or small lymphocyticlymphoma (SLL), or a myelodysplastic syndrome.

In some embodiments, the therapeutically effective amount of3-(4-amino-1-oxo-1,3 dihydro-isoindol-2-yl)piperidine-2,6-dione or apharmaceutically acceptable salt thereof, is between about 1 mg to about50 mg per day. In still other embodiments the therapeutically effectiveamount of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)piperidine-2,6-dione or a pharmaceuticallyacceptable salt thereof, is between about 1 mg to about 50 mg per day.While in still other embodiments the therapeutically effective amount of3-(4-amino-1-oxo-1,3 dihydro-isoindol-2-yl)piperidine-2,6-dione or apharmaceutically acceptable salt thereof, is between about 5 mg to about50 mg per day. And in still other embodiments the therapeuticallyeffective amount of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)piperidine-2,6-dione or a pharmaceuticallyacceptable salt thereof, is between about 1 mg to about 25 mg per day.

In some embodiments the therapeutically effective amount of 1-7F9 isbetween about 1 mg/kg to about 3 mg/kg per month. In still otherembodiments the therapeutically effective amount of 1-7F9 is betweenabout 1 mg/kg of the patient's body weight to about 3 mg/kg per month.

Still other aspects of the invention include kits for inducing celldeath, comprising: a biologically active amount of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)piperidine-2,6-dione or a pharmaceuticallyacceptable salt thereof; and a biologically active amount of an anti-KIRantibody or biologically active fragment thereof. In some embodimentsthe anti-KIR antibody in the kit is 1-7F9.

Yet other aspects of the invention include kits for inducing fortreating a disease, comprising: at least one therapeutically effectivedose of 3-(4-amino-1-oxo-1,3 dihydro-isoindol-2-yl)piperidine-2,6-dioneor a pharmaceutically acceptable salt thereof; and at least onetherapeutically effective dose of an anti-KIR antibody or biologicallyactive fragment thereof. In some embodiment the anti-KIR antibody in thekit is 1-7F9.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Graph illustrating the effect of different effector (E: NKcells) to tumor target (T) ratios (E:T ratios) on the percent ofspecific release.

FIG. 2. Schematic illustrating the interaction between NK cells and U266cells.

FIG. 3. Bar graph illustrating the effect of lenalidomide (10 μM) onenhancing killing of U266 cells by normal donor peripheral bloodmononuclear cells (PBMC) at different E:T ratios.

FIG. 4. Graph illustrating enhanced NK cell mediated cytotoxicity ofU266 human myeloma cells by the combination of 1-7F9 and lenalidomide.

FIG. 5. Assays measuring the effect of treating purified NK cells witheither lenalinomide in DMSO (light lines) or DMSO alone (dark lines) onthe expression levels of the NK cell receptors NKG2D, NKG2A, KIR2DL1/S1,and KIRDL2/L3.

FIG. 6. Graph illustrating the effect of pre-incubating 0266 targetcells with lenalidomide on their susceptibility to lysis by fresh donorNK cells at different E:T ratios.

FIG. 7. Results for a representative experiment showing thatlenalidomide increases the expression of activating ligands on targetcells.

FIG. 8. Bar graph summarizing the effect of lenalidomide on theexpression of activating ligands on U266 cells in at least 4 independentexperiments.

FIG. 9. Western blot analysis of cell lysates illustrating the effect of10 μmolL⁻¹ lenalidomide versus DMSO on the expression of phospho-p44/42(ERL1/2; Thr202/Tyr2040, pSHIP (Tyr1020). GAPDH is used as a proteinloading control.

DESCRIPTION

For the purposes of promoting an understanding of the principles of thenovel technology, reference will now be made to the preferredembodiments thereof, and specific language will be used to describe thesame. It will nevertheless be understood that no limitation of the scopeof the novel technology is thereby intended, such alterations,modifications, and further applications of the principles of the noveltechnology being contemplated as would normally occur to one skilled inthe art to which the novel technology relates.

As used herein, unless stated otherwise the term ‘about’ means plus orminus 10 percent, e.g. about 1 encompasses values ranging from 0.9 to1.1.

As used herein unless stated otherwise, the terms, ‘biologically activeamounts’ or ‘biologically active doses,’ mean amounts or doses capableof induce a noticeable effect on cells or cellular components aftereither a single dose or multiple doses of the compounds.

As used herein unless stated otherwise, the term, ‘therapeuticallyeffective amounts’ or ‘therapeutically effective doses,’ mean amounts ordoses capable of producing a beneficial effect on a human or animalafter either a single or multiple treatments with the compounds.

Tumor cells that express normal levels of MI-IC class 1 molecules may beresistant to killing by autologous NK cells because of inhibitorysignals mediated by the interaction of inhibitory KIR with MHC class Iligands.

The anti-KIR antibody 1-7F9 blocks the interaction of KIR and MHC classI molecules and enhances susceptibility to killing by autologous NKcells and is being developed for clinical anti-cancer therapy. Theanti-KIR antibodies used according to the invention reduce KIR mediatedinhibition of NK cells. Other anti-KIR antibodies may bind and block thesignaling activity of inhibitory KIRs including one or more members ofthe KIR2DL subfamilies. Inhibitory KIRs having two Ig domains (KIR2DL)recognize HLA-C allotypes: KIR2DL2 (formerly designated p58.2) and theclosely related, allelic gene product KIR2DL3 both recognize “group 1”HLA-C allotypes (including HLA-Cw1, -3, -7, and -8), whereas KIR2DL1(p58.1) recognizes “group 2” HLA-C allotypes (such as HLA-Cw2, -4, -5,and -6). The recognition by KIR2DL1 is dictated by the presence of a Lysresidue at position 80 of HLA-C alleles. KIR2DL2 and KIR2DL3 recognitionis dictated by the presence of an Asn residue at position 80 in HLA-C.Importantly, the great majority of HLA-C alleles have either an Asn or aLys residue at position 80. Therefore, KIR2DL1, -2, and -3 collectivelyrecognize essentially all HLA-C allotypes found in humans. Determiningwhether an antibody reduces MR mediated inhibition can be tested bystandard methods such as in a chromium assay with an NK cell cloneexpressing one or several KIRs, and a target cell expressing only oneHLA allotype recognized by one of the KIR of the NK clone and no otherHLA class I molecule recognized by the other KIRs on the NK clone, thelevel of cytotoxicity obtained with the antibody should be at least 60%preferably at least 70%, or more of the cytotoxicity obtained with acontrol antibody that blocks the interactions between KIR and HLA-C.WO2005003172, WO2005003168, and WO2005009465, each of which is hereinincorporated by reference in its entirety, describe KIR2DL1 andKIR2DL2/3 cross-reactive antibodies such as, e.g., DF200, 1-7F9, 1-4F1,1-6F5, and 1-6F1, and that DF200 and 1-7F9 enhance NK cell cytotoxicity.Other anti-KIR antibodies include GL183 and EB6 available from (BeckmanCoutler or Immunotech, France). Any of these antibodies or antibodiesthat compete with these for binding a MR polypeptides and block KIRsignaling can be used.

Doses of anti-KIR antibodies are described, for example, inWO/2008/084106 which is herein incorporated by reference in itsentirety. Optionally, the dose of anti-KIR antibody is in the range fromabout 0.0003 to about 3 mg/kg; from about 0.003 to about 3 mg/kg; fromabout 0.015 to about 3 mg/kg; from about 0.075 to about 3 mg/kg; fromabout 0.075 to about 3 mg/kg; from about 0.3 to about 3 mg/kg, and fromabout 1 to about 3 mg/kg. Exemplary doses are about 0.0003, about 0.003,about 0.015, about 0.075, about 0.3, about 1, and about 3 mg/kg.

Immunomodulatory agents (e.g., lenalidomide, thalidomide, and otheranalogues) enhance NK cell function and number in vitro and in vivo andhave significant activity against a variety of cancers through a numberof mechanisms. Given the ability of immunomodulatory agents to enhanceNK cells activity, they may work synergistically with anti-KIR antibodyto enhance immunological attack on cancer cells. The combination ofimmunomodulatory agents and anti-KIR may represent a novel immunotherapyapproach for a variety of different cancers.

The function of natural killer (NK) cells is regulated by inhibitory andactivating signals delivered by cell surface receptors. Interaction of‘self’ MHC class I molecules with inhibitory receptors expressed on thesurface of NK cells inhibits NK cell-mediated cytotoxicity againstautologous cancer cells. 1-7F9 is a human monoclonal antibody (mAb)directed against KIR2DL1, -2 and -3 receptors that blocks theirinteraction with HLA-C ligands, thereby breaking the NK cell's toleranceof autologous tumor cells. The mAb 1-7F9 is currently undergoingclinical investigation as a single agent in phase I trials in multiplemyeloma (MM) and acute myeloid leukemia.

For an additional discussion of the bio-synthesis, isolation,characterization and uses for the monoclonal antibody 1-7F9 and variousbioactive fragments, derivatives and formulations thereof the reader isdirected to various publications, including, but not limited to, U.S.application Ser. No. 11/324,356 filed on Jan. 3, 2006, which publishedon Dec. 14, 2006 as publication number US2006/0280740 a correctedversion of which was published on Jul. 30, 2009 as publication numberUS2009/0191213A9, each of which is herein incorporated by reference inits entirety as if each were separately incorporated by reference in itsentirety.

The compound lenalidomide is highly effective in the clinical treatmentof MM, and has also been shown to increase NK cell cytotoxicity invitro. This compound is available commercially and sold under the tradenames Revlimid® and Revimid®. For an additional discussion of thesynthesis, isolation, characterization and uses of lenalidomide and itsanalogs, derivatives, formulations, pharmaceutically acceptable saltsand bio-available crystal forms thereof, the reader is directed tovarious publications, including, but not limited to, U.S. Pat. No.5,635,517 filed on Jul. 24, 1996; U.S. Pat. No. 6,045,501 filed on Aug.28, 1998; U.S. Pat. No. 6,281,230 filed on Apr. 15, 2000; U.S. Pat. No.6,315,720 filed Oct. 23, 2000; U.S. Pat. No. 6,555,554 filed on Feb. 12,2001; U.S. Pat. No. 6,561,976 filed on Sep. 26, 2001; U.S. Pat. No.6,561,977 filed on Sep. 27, 2001; U.S. Pat. No. 6,755,784 filed on Mar.7, 2003, U.S. Pat. No. 6,908,432 filed on Jan. 22, 2004; U.S. Pat. No.7,119,106 filed Jan. 6, 2003, U.S. Pat. No. 7,189,740 filed Apr. 11,2003, and U.S. Pat. No. 7,465,800 filed Sep. 3, 2004, each of which isherein incorporated by reference in its entirety. Lenalidomide has beeninvestigated in a variety of conditions, including B-cell cancer (e.g.multiple myeloma (MM)), a leukemia, a lymphoma, chronic lymphocyticleukemia (CLL) or small lymphocytic lymphoma (SLL), myelodysplasticsyndromes (e.g. associated with the chromosome 5 abnormality).

The potential for synergy between lenalidomide and 1-7F9 against cancercells such as MM cells was investigated. Europium release cytotoxicityassays show that 1-7F9 blocking enhances NK cell mediated cytotoxicityagainst the relatively resistant MM U266 cells. U266 cells co-expressgroups 1 and 2 HLA-C class I ligands and are thus capable of inhibitingNK cells found in the majority of donors. The monoclonal antibody 1-7F9significantly enhanced natural cytotoxicity against U266 cells of bothpurified NK cells at Effector:Target (E:T) ratios of 10:1 or less, andalso of freshly isolated peripheral blood mononuclear cells (PBMC) atE:T ratios of 60:1 or less, from more than 10 random donors. Inaddition, treatment of PBMC with 10 μmolL⁻¹ lenalidomide for 72 hourswith or without interleukin (IL)-2 increased NK cell induced lysis ofU266 cells. Finally, the combination of lenalidomide and 1-7F9 enhancedNK cell mediated cytotoxicity against U266 cells beyond that observedwith each agent alone indicating a synergistic, or at least an additiveeffect.

Referring now to FIG. 1. Graph illustrating the effect of differenteffector (E: NK cells) to tumor target (T) ratios (E:T ratios) on thepercent of specific release (killing of tumor target cells measured inthe presence and absence of 30 μg ml⁻¹ of the antibody 1-7F9. 1-7F9enhances the cytotoxicity of NK cells against 0266. No Ab vs 1-7F9:10:1, p=0.0353; 5:1, p=0.0129; 2.5:1, p=0.0049; 1.25:1, p=0.0019.

Referring now to FIG. 2. Schematic illustrating the interaction betweenNK cells and U266 cells. This schematic illustrates that the MM cellline U266 expresses both groups 1 and group 2 MHC class molecules;HLA-A, HLA-B, and group 1 HLA-C ligands on the cell surface whichinhibit KIR2DL1 and KIR2D12/3 which are blocked by the anti-KIR antibody1-7F9. Briefly, KIR2DL2 and KIR2DL3 bind to HLA-C* 0304 and HLA-C* 0702and then block the NK cytotoxicity. 1-7F9 is monoclonal antibody againstKIR2DL2/3 that block the interaction of KIR2DL2/3 with HLA-Cw3 orsimilar allotypes then increases the NK cytotoxicity.

Referring now to FIG. 3, Effector cells: PBMC were treated with DMSO or10 μmolM-1 lenalidomide for 72 h without IL-2. The target cells wereU266 cells. Still referring to FIG. 3, a bar graph illustrating theeffect of lenalidomide (10 μM) on enhancing killing of U266 cells bynormal donor peripheral blood mononuclear cells (PBMC) at different E:Tratios as measured by the percent of specific release in a Europiumrelease cytotoxicity assay. Lysis of U266 target cells is measured inthe presence and absence of 10 μM lenalidomide. The enhancedcytotoxicity of PBMC against U266 by lenalidomide occurred withoutadding interleukin (IL)-2 to the culture. The total NK cell numbers wasessentially the same in the culture with DMSO treatment and in theculture with 10 μmol/L lenalidomide treatment. Accordingly, differencesin the number of NK cells does not account for why lenalidomide enhancesthe NK cytotoxicity.

Referring now to FIG. 4. Peripheral blood mononuclear cells (PBMC) wereobtained from normal donors following a Ficoll gradient separation.These cells were incubated for 72 hours in lenalidomide (10 μmolL⁻¹) orDMSO as control, and then used as effector cells against U266 targetcells in the presence or absence of 1-7F9 blocking antibody in Europiumrelease cytotoxicity assays. Cytotoxicity assays were performed in thepresence or absence of 30 μg/ml 1-7F9. In experiments investigating thecombination, PBMC were treated with lenalidomide (10 μmolL⁻¹) or DMSO(control) for 72 hours, and then used as effector cells in the presenceor absence of 1-7F9 (30 μgml⁻¹) in europium release assay. At an E:Tratio of 60:1, 30 μg/ml 1-7F9 augmented specific lysis of U266 cellsfrom 19±4% for DMSO-treated control PBMC to 32±2.2% (P=0.01).Lenalidomide (10 μmolL⁻¹) enhanced specific cell lysis to 27±1.3%relative to control (P0.03), while the combination of lenalidomide (10μmol/L) and 1-7F9 (30 μg/ml) resulted in specific cell lysis of 43±1.9%,which was significantly higher than lenalidomide alone (P=0.0003) or1-7F9 mAb alone (P=003) (FIG. 4).

Still referring to FIG. 4, the combination of lenalidomide and 1-7F9enhanced NK cell mediated cytotoxicity against U266 cells. At 60:1ratio:DMSO vs Lena, p=0.0343; DMSO vs 1-7F9, p=0.01; DMSO vs(Lena+1-7F9), p=0.0008; Lena vs Lena+1-7F9; 0.0003; 1-7F9 vs Lena+1-7F9,p=0.0031. At 30:1 ratio: DMSO vs Lena, p=0.0006; DMSO vs 1-7F9,p=0.0098; DMSO vs (Lena+1-7F9(, p=0.0002; Lena vs Lena+1-7F9, 0.0011;1-7F9 vs Lena+1-7F9, p=0.0019. These results show that the combinationof 1-7F9 anti-KIR mAb and lenalidomide have efficacy against other tumortypes where lenalidomide might also exhibit activity. Accordingly, thiscombination provides of a method for treating cancers such asnon-Hodgkin's lymphoma and the like.

Still referring to FIG. 4. As shown, U266 are relatively resistant to NKcell mediated killing (dark blue curve). 1-7F9 enhances the killing ofU266, as does pre-incubation of PBMC with lenalidomide. Treatment withlenalidomide enhanced cytotoxicity of NK cells against U266 cells.Similarly, the use of 1-7F9 blocking anti-KIR antibody facilitated thekilling of U266 cells. However, the combination of treatment of PBMCwith lenalidomide and the use of 1-7F9 acts synergistically to enhancekilling of U266 compared to either lenalidomide or 1-7F9 alone.

Referring now to FIG. 5. NK cells were purified from buffy coats ofnormal blood donors by immunomagnetic selection (CD3 depletion followedby CD56 selection) using the autoMACS (Milteny Biotec, Auburn, Calif.).Purified NK cells were cultured in RPMI medium with 10% human AB serumfor 72 hours in the presence or absence of lenalidomide. NK cells werethen assayed for receptor expression by flow-cytometry. As shown,lenalidomide has no effect on NK cell receptor expression indicatingthat its ability to enhance NK cell mediated lysis in combination with1-7F9 is not related to upregulation of activating receptors (NKG2D) ordownregulation of inhibitory receptors (NKG2A, KIR2DL1 or KIR2DL2/3).The levels of NK cells receptors expression were not significantlychanged by treatment with lenalidomide.

Referring now to FIG. 6. U266 cells were cultured in RPMI medium with10% human AB serum (complete medium) in the presence or absence oflenalidomide (5 □M) for 5 days, and then used as target cells inEuropium release cytotoxicity assays. As shown, U266 cells cultured inthe presence of lenalidomide (pink curve) were more susceptible to lysiscompared to U266 not exposed to lenalidomide (blue curve), indicatingthat lenalidomide may mediate its effect largely by enhancingsusceptibility of target cells to NK mediated lysis. These resultsindicate that lenalidomide enhances the susceptibility of myeloma celllines to NK cells in vitro.

Referring now to FIG. 7. Results for a representative experiment showingthat lenalidomide increases the expression of activating ligands ontarget cells. Briefly, U266 cells were cultured in complete medium inthe presence or absence of lenalidomide (5 μM) for 5 days. U266 cellswere then assayed by flow-cytometry for the level of expression of theactivating ligands MICA, MICA/B, DR4, DR5, ULBP1, ULBP2, PVR andNectin-2 (CD112) in U266 cells. As shown, lenalidomide enhanced theexpression of the activating ligands to varying extents (see below).

Referring now to FIG. 8. Bar graph summarizing the effect oflenalidomide on the expression of activating ligands on U266 cells in atleast 4 independent experiments. In this experiment U266 cells wereanalyzed as described as in the above. The data were analyzed using thepaired t-test. In this experiment lenalidomide is shown to significantlyincrease the expression of only MICA, DR4, ULBP2, and CD112 on U266cells. The data strongly suggest that lenalidomide enhances thesusceptibility of U266 cells to NK cell mediated killing by increasingthe expression of activating ligands. Consistent with these resultslenalidomide significantly increases the expression of MICA, ULBP2, DR4,and CD112 on U266 cells.

Referring now to FIG. 9. Western blot analysis was run on lysates ofcells treated with either DMSO or 10 μmol/L. The gels were probed withantibodies to phospho p44/42 (Erk1/2) Thr202/Tyr204); p-SHIP1 (Tyr1020)and GAPDH, the antibodies were obtained from Cell Signaling Technology(Danvers, Mass. U.S.A.). The various bands were quantified bydensitometry and the amounts of phospho-p44/42 (Erk1/2) Thr202/Tyr204)and p-SHIP1 (Tyr1020) were normalized to the amount of GAPDH measured ineach lane. The values determined for phospho-p44/42 (Erk1/2)Thr202/Tyr204)/GAPDH measured from cells treated with either DMSO or 10μmol/L lenalidomide were 1.00 and 0.98, respectfully. The valuesdetermined for P-SHIP1(Tyr1020)/GAPDH measured from cells treated witheither DMSO or 10 μmol/L lenalidomide were 1.00 and 1.76, respectfully.Still referring to FIG. 9, these results indicate that lenalidomide mayup-regulate the expression phospho-SHIP1 (Tyr1020) but it appears tohave little or no effect on phospho-p44/42 (ERK1/2) (Thr202/Tyr204)expression in NK cells.

As described herein the monoclonal antibody 1-7F9 enhances thecytotoxicity of NK cells against myeloma cells in vitro. The combinationof lenalidomide and 1-7F9 enhance NK cell mediated cytotoxicity againstU266 cells beyond the results observed with each agent alone indicatinga synergistic, or at least an additive effect. Lenalidomide increasesthe expression of activating NK cell receptor ligands on U266 cells andenhances the susceptibility of myeloma cell lines to NK cell basedcytotoxicity in vitro. Lenalidomide may affect intracellular signalingpathway in NK cells, e.g., up-regulating the phospho-SHIP1 (Tyr1020) inNK cells.

Materials and Methods

Effector cells: PBMC and NK cells were collected from random donors.Target cells: The myeloma (MM) cell line U266 co-expresses class I andII HLA-C ligands. The K562 cell line is negative for HLA class Imolecules and acts as negative control for 1-7F9 blocking experiment.

The Europium release cytotoxicity assay was performed according to theinstruction of DELFIA® EuTDA Cytotoxicity AD0116 kit (PerkinElmer).

While the novel technology has been illustrated and described in detailin the figures and foregoing description, the same is to be consideredas illustrative and not restrictive in character, it being understoodthat only the preferred embodiments have been shown and described andthat al changes and modifications that come within the spirit of thenovel technology are desired to be protected. As well, while the noveltechnology was illustrated using specific examples, theoreticalarguments, accounts, and illustrations, these illustrations and theaccompanying discussion should by no means be interpreted as limitingthe technology. All patents, patent applications, and references totexts, scientific treatises, publications, and the like referenced inthis application are incorporated herein by reference in their entirety.

I claim:
 1. A method of treating a patient having multiple myeloma,comprising administering to the patient a therapeutically effectiveamount of (a) 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)piperidine-2,6-dione or a pharmaceuticallyacceptable salt thereof, and (b) an antibody or a fragment thereof that(i) cross-reacts with KIR2DL1 and KIR2DL2/3, and (ii) enhances NK cellcytotoxicity.
 2. The method according to claim 1, wherein the patient isa human.
 3. The method according to claim 1, wherein the therapeuticallyeffective amount of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)piperidine-2,6-dione or a pharmaceuticallyacceptable salt thereof, is between about 1 mg to about 50 mg per day.4. The method according to claim 1, wherein the therapeuticallyeffective amount of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)piperidine-2,6-dione or a pharmaceuticallyacceptable salt thereof, is between about 5 mg to about 50 mg per day.5. The method according to claim 1, wherein the therapeuticallyeffective amount of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)piperidine-2,6-dione or a pharmaceuticallyacceptable salt thereof, is between about 1 mg to about 25 mg per day.6. The method according to claim 1, wherein the therapeuticallyeffective amount of said antibody or fragment thereof is between about 1mg/kg to about 3 mg/kg per month.
 7. A method of treating a patienthaving multiple myeloma, consisting essentially of administering to thepatient a therapeutically effective amount of (a) 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)piperidine-2,6-dione or a pharmaceuticallyacceptable salt thereof and (b) an antibody or a fragment thereof that(i) cross-reacts with KIR2DL1 and KIR2DL2/3, and (ii) enhances NK cellcytotoxicity.
 8. A kit for inducing death of myeloma cells, comprising:a biologically active amount of 3-(4-amino-1-oxo-1,3dihydro-isoindol-2-yl)piperidine-2,6-dione or a pharmaceuticallyacceptable salt thereof; and a biologically active amount of an antibodyor a fragment thereof that (i) cross-reacts with KIR2DL1 and KIR2DL2/3,and (ii) enhances NK cell cytotoxicity.
 9. The kit according to claim 8,wherein the antibody or fragment thereof comprises a light chainvariable region having the amino acid sequence set forth in SEQ ID NO:1and a heavy chain variable region having the amino acid sequence setforth in SEQ ID NO:2.
 10. A kit for treating multiple myeloma,comprising: at least one therapeutically effective dose of3-(4-amino-1-oxo-1,3 dihydro-isoindol-2-yl)piperidine-2,6-dione or apharmaceutically acceptable salt thereof; and at least onetherapeutically effective dose of an antibody or a fragment thereof that(i) cross-reacts with KIR2DL1 and KIR2DL2/3, and (ii) enhances NK cellcytotoxicity.
 11. The kit according to claim 10, wherein the KIR2DL1 andKIR2DL2/3 cross-reactive antibody or a fragment thereof that enhances NKcell cytotoxicity comprises a light chain variable region having theamino acid sequence set forth in SEQ ID NO:1 and a heavy chain variableregion having the amino acid sequence set forth in SEQ ID NO:2.
 12. Themethod according to claim 1, wherein said KIR2DL1 and KIR2DL2/3cross-reactive antibody or fragment thereof comprises a light chainvariable region having the amino acid sequence set forth in SEQ ID NO:1and a heavy chain variable region having the amino acid sequence setforth in SEQ ID NO:2.
 13. The method according to claim 7, wherein saidKIR2DL1 and KIR2DL2/3 cross-reactive antibody or fragment thereofcomprises a light chain variable region having the amino acid sequenceset forth in SEQ ID NO:1 and a heavy chain variable region having theamino acid sequence set forth in SEQ ID NO:2.
 14. The kit according toclaim 8, wherein the antibody or fragment thereof comprises a lightchain variable region having the amino acid sequence set forth in SEQ IDNO:1 and a heavy chain having the amino acid sequence set forth in SEQID NO:3.
 15. The kit according to claim 10, wherein the antibody orfragment thereof comprises a light chain variable region having theamino acid sequence set forth in SEQ ID NO:1 and a heavy chain havingthe amino acid sequence set forth in SEQ ID NO:3.
 16. The methodaccording to claim 1, wherein the antibody or fragment thereof comprisesa light chain variable region having the amino acid sequence set forthin SEQ ID NO:1 and a heavy chain having the amino acid sequence setforth in SEQ ID NO:3.
 17. The method according to claim 7, wherein theantibody or fragment thereof comprises a light chain variable regionhaving the amino acid sequence set forth in SEQ ID NO:1 and a heavychain having the amino acid sequence set forth in SEQ ID NO:3.